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The Generation of Red Cells from Stem Cells. Dave Anstee Director, NIHR Blood and Transplant Research Unit Red Cell Products University of Bristol The process Multipotential haematopoietic stem cell 106 CD34+ HPCs from a single apheresis cone, buffy coat or cord blood unit Proerythroblast Basophilic erythroblast 21 days Polychromatic erythroblast Orthochromatic erythroblast Mature reticulocytes – leucocyte filtration Reticulocyte Erythrocyte Cultures in spinner vessels Mature functional human reticulocytes can be generated in a liquid culture system from peripheral blood CD34+ cells. • α and ß Globins • Reversible O2 binding • Normal expression of blood group active proteins and cell surface antigens •Absence of T and Tn antigens • Deformability • Proteome Griffiths et al.Blood 2012;119(26):6296, Wilson et al.Mol Cell Proteomics 2016;15(6):1938 Reticulocytes reduce surface area and volume and remove residual organelles by expulsion of autophagic vesicles. Mankelow et al. Blood 2015; 126(15): 1831 Role of the Spleen . in Vesicle Removal 4 % cells with vesicles PS 3 BRIC163 BRIC155 2 Elevated levels of vesicles on circulating red cells in splenectomised patients suggests the spleen is important for their removal. 1 0 Control 1 Patient 1 Control 2 Patient 2 . Mankelow et al. Blood 2015;126(15):1831 . 3D Adult Cultured Reticulocytes remodel in vivo (mouse). 10 min 24 hours Becky Griffiths BUT • Need a sustainable supply of cultured red cells of desired blood group phenotype Generation of the First Adult Erythroid Progenitor Cell Lines Adult BM CD34+ D0 1o medium D1 D2-4 Transduce with Tet- 1o medium inducible HPV16 E6/E7 D5 Transfer to Expansion medium + doxycycline BEL-A Dif. d4 Dif. d10 Dif. d18 Filtered retics Adult Dif. d4 Dif. d14 Dif. d18 Filtered retics “Tom” Trakarnsanga Method of Kurita et al. Plos One 2013;8(3):e59890 Live imaging of filtered reticulocytes showing presence of autophagic vesicles PS PS BRIC163 BRIC155 Adult Peripheral Blood BELA Becky Griffiths BELA Reticulocytes in vivo (mouse) 10 min 3D 24 hours Becky Griffiths Why do we need cultured red cells? For patients with rare blood groups. For patients with Sickle Cell Disease • . Based on data from Dr.Fiona Regan New Technologies applied in Transfusion Medicine 2015 2000 1975 1945 Gene Editing Molecular Diagnostics Development of Monoclonal Antibody Technology Development of Antiglobulin test Genome editing BELA’s using CRISPR Number of cells Glycophorin A Knockdown Basigin Knockdown RHAG Knockout RhD and RhCE knockdown APC Fluorescence Intensity (arb. units) Wild type BEL-A IgG control CRISPR Knockdown Joe Hawksworth, Tim Satchwell and Ash Toye Autophagic Vesicles from Reticulocytes remain in the Blood of Patients with Sickle Cell Disease BRIC163 BRIC155 % cells with vesicles SCD patient PS 5 4 3 2 1 0 PS BRIC163 BRIC155 Mankelow T et al. Blood 2015;126(15):1831 Current Challenges • Induce maturation of cultured reticulocytes to erythrocytes in vitro. • Develop technology for scale up to therapeutic doses. Sabine Taylor Nicky Cogan Develop Technology for Scale Up - two approaches Functionalised Scaffolds Genetic Manipulation Use of synthetic scaffolds for ex vivo culture T75 with scaffold and 40ml expansion medium 1.6 liters in Spinner Flask Severn et al. Scientific Reports 2016;6:32149 Summary • Erythroid progenitor cell lines provide a sustainable supply of cultured reticulocytes with the same properties as those obtained from cultures of CD34+ cells. • Gene Editing of Blood Group Genes will allow the manufacture of cultured reticulocytes with different rare blood group phenotypes from a single founder erythroid progenitor cell line. • Further work is required to effect maturation of cultured reticulocytes to erythrocytes in vitro and to manipulate erythroid progenitor cell lines to maximise yield of reticulocytes but the technology required to do this is now available. The work is funded by National Institute of Health Research (NIHR) NHS Blood and Transplant Wellcome Trust Key Collaborators: Yukio Nakamura, Riken Institute,Tokyo Sara Trompeter,UCLH, London NIHR Blood and Transplant Research Unit University of Bristol 2016